Intracerebroventricular enzyme replacement therapy with glycosylation-independent lysosomal targeted NAGLU leads to widespread enzymatic activity, reduction of lysosomal storage and of secondary defects in brain of mice with Sanfilippo syndrome type B

Treatment for mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome type B) is hampered because recombinant alpha-N-acetylglucosaminidase (NAGLU) contains little/no mannose 6-phosphate. We developed a fusion protein of insulin-like growth factor 2 (IGF2) and NAGLU (rhNAGLU-IGF2). IGF2 is a natural ligand of the mannose-6phosphate receptor, and thus provides glycosylation-independent lysosomal targeting. Purified rhNAGLU-IGF2 is active in biochemical and cell-based assays (see Abstract: Aoyagi-Scharber, et al.). To evaluate rhNAGLU-IGF2 in vivo, adult (16 wk) MPS IIIB mice were catheterized into the left ventricle and given 4 twice-weekly injections of vehicle or rhNAGLU-IGF2 (100 μg). Mice were harvested 1d, 7d, 14d, and 28d after the last injection (n= 4-8 per group). Vehicle-treated heterozygotes were used as controls. Forebrain NAGLU activity ranged from 100x (1d, p b 0.001), 400x (7d, p = 0.001), 30x (14d, p = 0.9), and 130x fold (28d, p = 0.002) in rhNAGLU-IGF2 treated mice vs. vehicle-treated heterozygotes. Hexosaminidase activity was reduced by 26% (1d), 44% (7d), 51% (14d), and 57% (28d) in rhNAGLU-IGF2 treated vs vehicle-treated MPS IIIB mice (p b 0.001). Brain heparan sulfate was reduced from 228 pmol/ 5 μg brain (in vehicle-treated MPS IIIB) to 79.3 (1d), 24.8 (7d), 20.8 (14d), 17.4 (28d) in rhNAGLU-IGF2-treatedMPS IIIBmice (p b 0.001), vs 10.5 in vehicle-treated heterozygotes. In multiple brain regions, robust neuronal uptake was evident by immunostaining with NAGLU antibody and reduced LAMP1 signal with rhNAGLU-IGF2 treatment. In a separate experiment using 8 week-old mice, secondary accumulations were measured 1d after the last injection. Neuronal accumulations of SCMAS, glypican 5, amyloid beta and P-GSK3 beta in themedial entorhinal cortex and of P-tau in the dentate gyrus were reduced to the control level of these proteins. Accumulation of CD68 in activated microglia in the somatosensory cortex was reduced about half-way to control. In summary, we achieved broad distribution of NAGLU and improvement in neuropathology with short-term intraventricular rhNAGLU-IGF2. Glycosylation-independent lysosomal targeting may permit development of superior therapy for Sanfilippo syndrome type B. Support from NIH/NINDS 1R21NS078314-01A1 and BioMarin Pharmaceutical Inc.